Analysis of Efflux Pump System and Other Drug Resistance Related Gene Mutations in Tigecycline-Resistant Acinetobacter baumannii

Background . The isolation of tigecycline-resistant Acinetobacter baumannii in recent years has brought great di ﬃ culties to clinical prevention and treatment. Purpose . To explore the e ﬀ ect of e ﬄ ux pump system and other resistance related gene mutations on tigecycline resistance in Acinetobacter baumannii . Methods . Fluorescence quantitative PCR was used to detect the expression levels of major e ﬄ ux pump genes ( adeB , adeJ , and adeG ) in extensive drug-resistant Acinetobacter baumannii . The minimum inhibitory concentration (MIC) of tigecycline was detected by the broth microdilution testing and e ﬄ ux pump inhibition experiment to assess the role of e ﬄ ux pump in tigecycline resistance of Acinetobacter baumannii . E ﬄ ux pump regulatory genes ( adeR and adeS ) and tigecycline resistance related genes ( rpsJ , trm , and plsC ) were ampli ﬁ ed by PCR and sequenced. By sequence alignment, tigecycline sensitive and tigecycline-insensitive Acinetobacter baumannii were compared with standard strains to analyze the presence of mutations in these genes. Results . The relative expression of adeB in the tigecycline-insensitive Acinetobacter baumannii was signi ﬁ cantly higher than that in the tigecycline sensitive Acinetobacter baumannii (114.70 (89.53-157.43) vs 86.12 (27.23-129.34), P = 0 : 025 ). When e ﬄ ux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added, the percentage of tigecycline-insensitive Acinetobacter baumannii with tigecycline MIC decreased was signi ﬁ cantly higher than that of tigecycline-sensitive Acinetobacter baumannii (10/13 (76.9%) vs 26/59 (44.1%)), P = 0 : 032 ); the relative expression of adeB in the MIC decreased group was signi ﬁ cantly higher than that in the MIC unchanged group (110.29 (63.62-147.15) vs 50.06 (26.10-122.59), P = 0 : 02 ); The relative expression levels of e ﬄ ux pumps adeG and adeJ did not increase signi ﬁ cantly, and there was no signi ﬁ cant di ﬀ erence between these groups. One adeR point mutation (Gly232Ala) and eight adeS point mutations (Ala97Thr, Leu105Phe, Leu172Pro, Arg195Gln, Gln203Leu, Tyr303Phe, Lys315Asn, Gly319Ser) were newly detected. Consistent mutations in trm and plsC genes were detected in both tigecycline-insensitive and tigecycline-sensitive Acinetobacter baumannii , but no mutation in rpsJ gene was detected in them


Introduction
Acinetobacter baumannii (AB) is a nonmotile, oxidase-negative, nonfermenting sugar Gram-negative bacillus [1]. It is highly environmentally adaptable and can survive up to 5 months on dry solid surfaces due to biological properties such as low nutrient requirements, ability to survive in most temperature and pH environments, high resistance to disinfectants, and ease of biofilm formation on nonliving surfaces [2]. At the same time, its intrinsic resistance and its ability to rapidly upregulate endogenous resistance mechanisms or actively acquire exogenous resistance genes allow it to evolve relatively quickly into a multiresistant bacterium with resistance to multiple antibiotics [3]. As a result, Acinetobacter baumannii is widely present in health care settings and difficult to eradicate. Extensive drug-resistant Acinetobacter baumannii (XDR AB) refers to insusceptibility to all classes of antimicrobial drugs except colistin and tigecycline [4,5], and several studies have shown that drug-resistant Acinetobacter baumannii infections have been shown to have a high mortality rate, especially for carbapenem-resistant Acinetobacter baumannii (CRAB) infections, which can range from 35% to 61% [6][7][8][9][10][11]. Tigecycline (TGC) is currently regarded as an effective drug against CRAB or XDR-AB [12,13], however, with the gradual application of tigecycline in clinical practice, tigecycline resistant Acinetobacter baumannii also emerged in recent years [14][15][16].
The mechanism of tigecycline resistance production by Acinetobacter baumannii is a hot topic of current interest, and existing studies suggest that overexpression of the efflux pump system plays a major role in multidrug resistant bacteria [17][18][19]. Closely related to Acinetobacter baumannii tigecycline is the RND family, which contains three major systems: adeABC, adeFGH, and adeIJK [20]. However, the degree of correlation between overexpression of each system and tigecycline resistance levels may vary, and whether the efflux pump system can play an important role in altering tigecycline resistance levels in clinical strains in which multiple resistance mechanisms may coexist, which issues are still controversial [21][22][23]. Meanwhile, it has been reported that the variation of adeRS two-component system changed the sensitivity of Acinetobacter baumannii to tigecycline by regulating the expression of efflux pump system [24,25]. Moreover, recent studies have identified mutations in tigecycline drug target genes or membrane permeabilityrelated genes in some strains without efflux pump overexpression, such as trm, plsC, and rpsJ genes [26][27][28], and the prevalence of these resistance genes in clinical drugresistant strains has been less studied so far. Therefore, we explored the role of the above mechanisms in tigecyclineresistant Acinetobacter baumannii in our hospital.  [29]. The efflux pump inhibition test is the drug sensitivity test of TGC combined with CCCP efflux pump inhibitor (10ug/ml). The breakpoint of tigecycline sensitivity is based on the FDA standard: MIC≤2ug/ml is sensitive and MIC≥8ug/ml is resistant. Escherichia coli ATCC25922 were used as the drug-susceptible quality control bacteria, and Acinetobacter baumannii standard strain ATCC17978 was used as the control bacteria.

Materials and Methods
2.3. RNA Extraction and cDNA Synthesis. Extraction of bacterial total RNA was referred to the recommended steps of TaKaRa's RNAiso Plus (No. 9108/9109) and cDNA synthesis (reverse transcription) was performed according to the steps recommended by Transcriptor cDNA Synth. Kit 1 (No. 04 897 030 001).

2.4.
Real-Time PCR. The standard strain of Acinetobacter baumannii ATCC17978 was used the control strain, and the mRNA expression of the rpoB reference gene of each target strain and the three efflux pump systems adeB, adeG, and adeJ of the RND family were detected, and the relative expression of these three efflux pump genes was calculated. Specific steps were as follows: Synthesis of primer sequences: The primer sequences of target genes adeB, adeG, adeJ, and rpoB internal reference genes are shown in Table 1, and the synthesis was commissioned by BGI (Beijing Liuhe) Co.Ltd.
Reaction system and conditions: Follow the steps recommended by LightCycler 480 SYBR Green I Master.
Result data processing: Both the amplification curve and the dissolution curve were single-peak curves, indicating that Computational and Mathematical Methods in Medicine the product amplification repeatability and specificity were good, and the qualified test data was collected for calculation. mRNA relative expression ðREÞ = 2 −△△ Ct, where △△ Ct = (Ct Target-Ct rpoB) target strain-(Ct Target -Ct rpoB) control strain.

Detection of Efflux Pump Regulator Gene adeRS and
Drug Resistance Related Genes plsC, Trm and rpsJ. Preparation of bacterial genomic DNA template is as follows: scrape a ring of fresh monoclonal bacteria cultured on LB agar medium overnight, resuspend in sterilized water, place in a 100°C water bath for 10 min, and collect the supernatant by centrifugation as the strain genomic DNA template. Synthesis of primer sequences is as follows: the primer sequences of regulatory genes adeR, adeS, and drug resistance-related genes plsC, trm, and rpsJ are shown in Table 2, and the synthesis was commissioned by BGI (Beijing Liuhe) Co.Ltd.
The target gene amplification was performed in the Applied Biosystems PCR system (Veriti Thermal Cycler) with Golden DNA polymerase (Golden Easy PCR System KT221, TIANGEN BIOTECH (BEIJING), China).
The amplification conditions of the regulated genes adeR and adeS are shown as follows. The amplification conditions included 35 cycles of amplification under the following conditions: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealed at 54°C for 30 s, extend at 72°C for 2 min, and a final extension at 72°C for 5 min.
The amplification conditions of drug resistance-related genes plsC, trm, and rpsJ were shown as follows. Amplification was carried out with the following thermal cycling conditions: 5 min at 94°C and 35 cycles of amplification consisting of 30 s at 94°C, 30 s at 58/56/54°C (for plsC/trm/ rpsJ, respectively), and 1 min at 72°C, with 5 min at 72°C for the final extension.
2.6. PCR Product Electrophoresis and Gel Imaging. Recovery, purification, and sequencing of PCR products by electrophoresis and gel cutting, BGI (Beijing Liuhe) Co., Ltd. was entrusted to carry out electrophoresis, gel cutting, recovery, and purification of PCR products, and the Sanger method was used for two-way detection and splicing.
Gene sequence alignment submitted the spliced gene sequence fragments to the PubMed website for BLAST alignment and annotated the unknown sequence fragments. Using the adeR, adeS, plsC, trm, and rpsJ genes in the complete genome of Acinetobacter baumannii standard strain ATCC17978 (NZ_CP018664.1) as the reference sequence, Bioedit and SnapGene software were used to perform multiple sequence alignment, translation, and mutation type of each spliced sequence identification.

Statistical
Methods. The database was established using SPSS version 21.0 software package and statistical processing was performed. Normally distributed measurement data were described as mean ± standard deviation ( x ± SD), and comparisons were made using independent samples t-test; nonnormally distributed measurement data were described as median (25% quantile~75% quantile); and described and compared using rank sum test. P < 0:05 difference was statistically significant. Graphs were drawn using GraphPad Prism 7.

Drug Susceptibility Test and Efflux Pump Inhibition Test.
The distribution of tigecycline MIC of 72 extensive drugresistant Acinetobacter baumannii strains was shown in Figure 1, and the range of MIC was distributed between 16-0.5 ug/ml. According to the FDA tigecycline susceptibility criteria (MIC≥8 ug/ml as resistant, MIC≤2 ug/ml as sensitive), these Acinetobacter baumannii were divided into two groups: tigecycline sensitive extensive drug-resistant Acinetobacter baumannii (TS-XDR AB) group, which tigecycline MIC was less than or equal to 2 ug/ml, and tigecycline insensitive extensive drug-resistant Acinetobacter baumannii (TIS-XDR AB) group, which tigecycline MIC was greater than 2ug/ml. Among them, there were 59 strains (81.9%) in TS-XDR AB group and 13 strains (18.1%) in TIS-XDR AB group. The number of MIC 2 and 1 strains was relatively high, with 24 (33.3%) and 28 (38.9%) strains, respectively. This part of the results showed that this TIS-XDR AB accounted for only a small proportion of XDR AB, but they could indeed be isolated from clinical patients, and even

Computational and Mathematical Methods in Medicine
individual TIS-XDR AB developed high levels of resistance to tigecycline.
When the efflux pump inhibitor CCCP was added, the statistics of the MIC decreased of tigecycline in Acinetobacter baumannii were shown in Table 3 and Figure 2. Among them, the tigecycline MIC of 36 strains decreased by 2-8 times, accounting for 50%, 26 strains (44.1%) were TS-XDR AB, and 10 strains (76.9%) were TIS-XDR AB. The proportion of TIS-XDR AB with tigecycline MIC decreased was higher than that in TS-XDR AB group, which was statistically significant (P = 0:032). That is, the tigecycline MIC decreased of the TIS-XDR AB group was inhibited by the efflux pump was significantly higher than the TS-XDR AB group. The tigecycline MIC decreased by more than 2 times, which significantly affected by CCCP, including 6 strains, accounting for about 8.3%, of which 4 strains were TIS-XDR AB, and 2 strains were TIS-XDR AB. This part of the results showed that efflux pump inhibitors play a more important role in reducing tigecycline sensitivity in TIS-XDR AB than in TS-XDR AB.

Analysis of the Relative Expression of Efflux Pump System
Detected by Real-Time Quantitative PCR. The relative expression (RE) of three main efflux pump genes was different between the TS-XDR AB group and the TIS-XDR AB group. Statistical analysis, the results are shown in Figure 3.
There is a significant difference in the relative expression of adeB gene between these two groups (P = 0:025). The relative expression of adeB in the TIS-XDR AB group is significantly higher than that in the TS-XDR AB group. However, there was no significant difference in the relative expression of adeG and adeJ genes between these two groups. This part of the results showed that adeB was significantly overexpressed in TIS-XDR AB compared with TS-XDR AB.

Analysis of the Relationship between the Relative
Expression of the Efflux Pump System and the Decrease in Tigecycline Sensitivity. Grouping according to the efflux pump inhibition experiment, the RE of three major efflux pump genes was statistically analyzed between the tigecyc-line MIC decreased group and the tigecycline MIC no changed group, and the results were shown in Figure 4.
It can be seen that there was a significant difference in the RE of adeB gene between these two groups; that is, the RE of adeB in the tigecycline MIC decreased group is significantly higher than that in the tigecycline MIC no changed group. However, there was no significant difference in the RE of adeG and adeJ genes between these two groups. This part of the results showed that the overexpression of adeB was closely related to the reduction of tigecycline resistance in XDR AB by efflux pump inhibitors.

Amplification and Sequence Alignment of Efflux Pump
Regulator Genes adeR and adeS and Drug Resistance-Related Genes plsC, Trm and rpsJ. The efflux pump regulation genes (adeR and adeS) and tigecycline resistance related genes (plsC, trm, and rpsJ) of 13 tigecycline insensitive extensive drugresistant Acinetobacter baumannii were detected, and 4 tigecycline sensitive extensive drug-resistant Acinetobacter baumannii were used as controls. The tigecycline MIC of these Acinetobacter baumannii were shown in Tables 4.
The expected amplified fragments of adeR and adeS genes are about 800 bp and 1200 bp, and most strains can amplify the corresponding specific target fragments. The    electropherogram of some strains were shown in Figure 5. Among them, the target fragments of the adeS gene amplified by the four strains 587, 576, 579, and 594 were significantly larger than expected, and the bands were about 2500 bp in Figure 5(b), suggesting that these genes may have inserted sequences. The expected amplified fragments of plsC, trm, and rpsJ genes are about 1400 bp, 1200 bp, and 300 bp, and each strain can amplify the corresponding specific purpose fragment, the electropherogram of PCR products of some strains were shown in Figure 6.
These 17 extensive drug-resistant Acinetobacter baumannii have detected mutations in adeR and adeS genes, including substitution, insertion mutation, and deletion mutations. The nonsynonymous mutations of each strain were shown in Table 5. It can be seen in the table that there were 5-6 missense mutations in the adeS gene of each strain, but only 2-3 missense mutations in adeR. Among them, the adeR of 511S, 576, and 594 strains had single-base insertions at different sites (Insert652A, Insert718G, Insert32C) resulting in nonsense mutation which is the early appearance of termination codons (229 Stop, 246 Stop, 13 Stop). The translationally synthesized peptide chain was shortened. In addition, the insertion sequence ISAba1 appeared at the 3′ end of the adeS of strains 576, 579, 587, and 594, which would affect the transcription of downstream genes. This part of the results showed that several nonsynonymous mutations were detected in adeR and adeS genes.

Computational and Mathematical Methods in Medicine
These 17 extensive drug-resistant Acinetobacter baumannii were found to contain mutations in plsC, trm genes, including substitution, insertion mutation, and deletion mutation. The nonsynonymous mutations of each strain were shown in Table 5. No mutation was found in the rpsJ. There was a missense mutation (CAA592AAA) and a terminator codon mutation (TAA925AAA) in the plsC gene. There were insertions or deletions bases at different sites (Insert29A, Dele-tion51A, Deletion267A) in the trm gene, resulting in nonsense mutation which is the early appearance of termination codons (16 Stop,37 Stop, 92 Stop), and the peptide chain synthesized by translation of the gene was shortened. This part of the results showed that nonsynonymous mutations were detected in both plsC and trm but not in rpsJ.

Discussion
The efflux pump mechanism is an important mechanism for the resistance of Acinetobacter baumannii tigecycline. The RND family is the first reported and widely recognized efflux pump family, including AdeABC, AdeFGH, and AdeIJK. Among them, overexpression of adeABC efflux pump system plays the largest role in drug resistance [30][31][32]. Efflux pump inhibitors restore sensitivity to the drug by inhibiting the active efflux of the Acinetobacter baumannii efflux pump.
In this study, the relative expression levels of the multidrug transporter genes adeB, adeG, and adeJ in the efflux pump systems adeABC, adeFGH, and adeIJK of 72 XDR-Aba strains were detected, and it was found that the relative expression levels of adeB were significantly increased. Moreover, the relative expression of TIS group was significantly higher than that of TS-XDR group, suggesting that overexpression of adeB was closely related to tigecycline resistance in Acinetobacter baumannii, and overexpression of adeB was the main factor reducing the sensitivity of tigecycline in Acinetobacter baumannii. While the relative expression of adeG and adeJ did not increase significantly, and there was no significant difference between the two groups, suggesting that the expression  Figure 5: Electropherogram of efflux pump regulation genes adeR and adeS of Acinetobacter baumannii ((a) adeR gene, 1-9 were 583, 587, 588, 594, 576, 579, 590S, 591, ATCC17978; (b) adeS gene, 1-9 were 583, 587, 588, 594, 576, 579, 590S, 591, respectively, of which 587, 594, 576, 579 target fragments were about 2500 bp, larger than expected fragments, suggesting that there may be an insertion sequence.).  6 Computational and Mathematical Methods in Medicine of these two efflux pump systems is not closely related to the tigecycline resistance of Acinetobacter baumannii, and their overexpression is not a cause of reduced tigecycline susceptibility in Acinetobacter baumannii. These results are consistent with other reports [21,33,34]. The efflux pump inhibition test also confirmed the above inference that after the addition of CCCP, the relative expression of adeB in the tigecycline MIC decreased group is significantly higher than that in the tigecycline MIC no changed group, suggesting that overexpression of adeB is closely related to the decreased tigecycline sensitivity induced by the inhibition of efflux pump, and overexpression of adeB is the main factor that efflux pump inhibitors reduce the tigecycline sensitivity of clinical Acinetobacter baumannii. While the relative expression of adeG and adeJ did not increase significantly, and there was no significant difference between the two groups, suggesting that the expression of these two efflux pump systems is not closely related to the decrease of tigecycline sensitivity due to the inhibition of efflux pumps, and their overexpression is not the reason that efflux pump inhibitors reduce tigecycline sensitivity in Acinetobacter baumannii. 50% (36 strains) of the strains showed a 2-8 fold decrease in MIC, suggesting that the active efflux of the efflux pump can increase the tigecycline MIC of most strains. However, only 8.3% (6 strains) of the strains had a greater than 2-fold decrease in MIC, of which 4 were non-susceptible strains, and the proportion of non-susceptible strains was 30.8% (4/13 strains), suggesting that the role of the efflux pump in affecting tigecycline MIC is limited. It is possible that multiple resistance mechanisms may combine to influence the sensitivity of tigecycline. In conclusion, although the efflux pump mechanism may play a limited role, the efflux pump system adeABC was widely present in Acinetobacter baumannii [35,36], which is a fundamental factor for increasing the level of tigecycline resistance, and is even more critical in the resistance mechanism of a small number of strains.
Overexpression of the adeABC efflux pump system is regulated by the two-component regulatory gene adeRS, and mutations in either the adeR or adeS genes may lead to adeABC expression [37][38][39]. adeB expression was significantly increased in all 13 tigecycline-insensitive strains, and detection of adeR and adeS genes in these strains revealed by comparison that they had 2-7 nonsynonymous mutations. The adeS gene was more prone to mutations than the adeR gene, a phenomenon in agreement with the studies of Montana S et al. [40] and Hammerstrom Troy G et al. [41], suggesting that adeS has a higher degree of genetic variation and plays a more active role in the regulation of adeABC overexpression. Four nonsynonymous point mutations (Val120Ile, Ala136Val, Gly232Ala, Asp20Asn) and one insertional mutation (Insert32C) were found in adeR gene, and 12 nonsynonymous point mutations (Ala94Val, Ala97Thr, Leu105Phe, Leu172Pro, Gly186Val Arg195Gln, Gln203Leu, Asn268His, Tyr303Phe, Lys315Asn, Gly319Ser, Val348Ile) and one insertional mutation (ISAba1 inserted at the end of adeS) were found in adeS gene. In addition to three adeR point mutations (Val120Ile, Ala136Val,    Asp20Asn) and four adeS point mutations (Ala94Val, Gly186Val, Asn268His, Val348Ile) and the insertion of ISAba1 mutation in adeS were reported to be associated with adeABC overexpression in previous studies [21,36,42,43]; the remaining one adeR point mutation (Gly232Ala) and eight adeS point mutations (Ala97Thr, Leu105Phe, Leu172-Pro, Arg195Gln, Gln203Leu, Tyr303Phe, Lys315Asn, Gly319Ser) were newly detected in this study, and these mutations may lead to elevated expression of adeABC, but their specific effects remain to be experimentally verified. In addition to the efflux pump mechanism, some studies in recent years have also reported that mutations in the trm, rpsJ and plsC genes lead to increased tigecycline resistance [26][27][28]. Therefore, this study detected the sequences of these genes in 13 tigecycline-insensitive strains and 4 tigecycline-sensitive strains, to understand the resistance mechanism of tigecycline-insensitive Acinetobacter baumannii in our hospital. Trm gene detection found that 17 strains all had single base deletions or insertions (Insert29A, Dele-tion51A, Deletion267A) at different sites, resulting in nonsense mutation which is the early appearance of stop codons (16 Stop,37 Stop, 92 Stop) and shortened synthesis. However, 4 of them were tigecycline-sensitive strains with MICs ranging from 0.25 to 1 ug/ml, so it was speculated that trm deletion could not significantly improve the resistance of Acinetobacter baumannii to tigecycline. This result is different from previous reports that the study compared the whole gene sequence of the tigecycline-insensitive mutant strain and its parental strain by genome sequencing, and found that the trm deletion mutation was the only difference between the two. It is speculated that the trm gene deletion leads to the decrease of tigecycline sensitivity. Chen Q et al. [27] also found a 35 bp nucleotide deletion in the trm gene of the drug-resistant mutant strain by genome sequencing. The wild-type trm gene complementation experiment restored the mutant strain's sensitivity to tigecycline, confirming that the trm gene mutation is one of the mechanisms of tigecycline resistance in Acinetobacter baumannii. Based on the above findings, we believe that trm gene deletion leads to tigecycline resistance is still controversial, and more studies are needed to clarify. plsC gene detection found that non-synonymous point mutations (Gln198Lys) and terminator codon mutations (Stop309Lys) were also present in tigecycline-sensitive Acinetobacter baumannii, suggesting that these mutations could not lead to tigecycline resistance. Although the study by Li X et al. [28] confirmed that the frameshift mutation of the plsC gene caused the synthetic shortened peptide chain leading to tigecycline resistance, this study did not detect the plsC gene deletion mutation. Therefore, the plsC gene deletion mutation is not the cause of the tigecycline resistance in Acinetobacter baumannii in our hospital. The rpsJ gene detection found that 13 strains did not find any mutation points, indicating that the gene is not related to tigecycline resistance in these strains.
In summary, our study revealed that the expression of adeABC efflux pump in extensive drug-resistant Acinetobacter baumannii was significantly increased, and the increase was more significant in tigecycline insensitive strains. The overexpression of adeABC was closely related to tigecycline resistance, and the mechanism of efflux pump was the cause of tigecycline resistance. It is a common and important mechanism, but only this mechanism has a limited impact, and most strains may have multiple resistance mechanisms that eventually lead to an increase of tigecycline resistance. AdeABC overexpression is the result of adeRS gene mutation, 1 adeR and 8 adeS point mutations have not been reported in previous studies, and the relationship between these mutant genes and adeABC overexpression remains to be explored. trm, plsC, and rpsJ genes have nothing to do with the resistance of Acinetobacter baumannii tigecycline in our hospital. The effect of trm, plsC, and rpsJ gene mutations on the development of tigecycline resistance in Acinetobacter baumannii remains unclear. Besides the pump mechanism, there might be other resistance mechanisms.

Data Availability
The datasets during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.